Search

Background & Aims Pancreatic ductal adenocarcinoma (PDA) initiation and progression are accompanied by an immunosuppressive inflammatory response. Here, we evaluated the immunomodulatory role of chemosensory signaling in metaplastic tuft cells (MTCs) by analyzing the role of GNAT3, a gustatory pathway G-protein expressed by MTCs, during PDA progression. Methods Gnat3-null (Gnat3-/-) mice were crossbred with animals harboring a Cre-inducible KrasLSL-G12D/+ allele with either Ptf1aCre/+ (KC) or tamoxifen-inducible Ptf1aCreERT/+ (KCERT) mice to drive oncogenic KRAS expression in the pancreas. Ex vivo organoid conditioned medium generated from KC and Gnat3-/-;KC acinar cells was analyzed for cytokine secretion. Experimental pancreatitis was induced in KCERT and Gnat3-/-;KCERT mice to accelerate tumorigenesis, followed by analysis using mass cytometry and single-cell RNA sequencing. To study PDA progression, KC and Gnat3-/-;KC mice were aged to morbidity or 52 weeks. Results Ablation of Gnat3 in KC organoids increased release of tumor-promoting cytokines in conditioned media, including CXCL1 and CXCL2. Analysis of Gnat3-/-;KCERT pancreata found altered expression of immunomodulatory genes in Cxcr2 expressing myeloid-derived suppressor cells (MDSCs) and an increased number of granulocytic MDSCs, a subset of tumor promoting MDSCs. Importantly, expression levels of CXCL1 and CXCL2, known ligands for CXCR2, were also elevated in Gnat3-/-;KCERT pancreata. Consistent with the tumor-promoting role of MDSCs, aged Gnat3-/-;KC mice progressed more rapidly to metastatic carcinoma compared with KC controls. Conclusions Compromised gustatory sensing, achieved by Gnat3 ablation, enhanced the CXCL1/2–CXCR2 axis to alter the MDSC population and promoted the progression of metastatic PDA., Graphical abstract

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDA) restricts vascularization and, consequently, access to blood-derived nutrients and oxygen, which impacts tumor growth. Intracellular redox imbalance is another restraint on cellular proliferation, yet it is unknown if the TME contributes to the maintenance of redox homeostasis in PDA cells. Here, we demonstrate that the loss of mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2), a component in the malate-aspartate shuttle, disturbs redox homeostasis and halts proliferation of PDA cells in vitro. In contrast, GOT2 inhibition has no effect on in vivo tumor growth or tumorigenesis in an autochthonous model. We propose that this discrepancy is explained by heterocellular pyruvate exchange from the TME, including from cancer associated fibroblasts. More broadly, pyruvate similarly confers resistance to inhibitors of mitochondrial respiration. Genetic or pharmacologic inhibition of pyruvate uptake or metabolism abrogated pyruvate-mediated alleviation of reductive stress from NADH buildup. In sum, this work describes a potential resistance mechanism mediated by metabolic crosstalk within the pancreatic TME. These findings have important implications for metabolic treatment strategies since several mitochondrial inhibitors are currently in clinical trials for PDA and other cancers.

Pancreatic ductal adenocarcinoma (PDA) is the third most common cause of cancer death in the US with a 9% 5-year survival rate. PDA is characterized by a robust stromal reaction, a large portion of which is composed of infiltrating immune cells and supported by fibroblasts. This fibroinflammatory response promotes both the initiation and progression of PDA. Key to this progression is the crosstalk between the tumor epithelium and immune compartment. Metaplastic tuft cells (MTCs) are a subset of the neoplastic epithelium derived by the transdifferentiation of acinar cells. Tuft cells have been characterized to have a “sense-and-respond” role in luminal surfaces throughout the body, using the taste chemosensory system to promote an inflammatory response in infected and damaged tissues. Among the pancreatic epithelium, MTCs uniquely express components of the gustation sensory system including taste receptors, TRPM5 and αGustducin, as well as an array of inflammatory cytokines, suggesting they are capable of promoting an immune response to extracellular challenges. To explore this hypothesis, we ablated αGustducin, a key protein in the chemosensory cascade, in a model of pancreatitis-associated tumorigenesis. We found that neoplasia in the GNAT3-/-; KrasG12D; PTF1aCreERT mice initiates and progresses more rapidly, a difference associated with alteration in immune cell subtypes. To further quantitate changes in the immune response, we performed mass cytometry (CyTOF), as a way to explore changes in cell number and type, and single-cell sequencing, to more deeply probe the unique gene signatures of cells within the tumor stroma. These large-scale analyses suggest substantial changes in the number of T-cell and myeloid cell populations, as well as alterations of fibroblasts and myeloid cell polarity. We conclude that MTCs use chemosensory signaling to promote the formation of a tumor-suppressive microenvironment. Citation Format: Megan T. Hoffman, Stephanie The, Daniel J. Salas-Escabillas, Kathleen E. DelGiorno, Filip Bednar, Robert F. Margolskee, Arvind Rao, Howard C. Crawford. The role of metaplastic tuft cell chemosensory signaling in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A19.

Intratumoral phenotypic heterogeneity has been described in many tumor types, where it can contribute to drug resistance and disease recurrence. We analyzed ductal and neuroendocrine markers in pancreatic ductal adenocarcinoma, revealing heterogeneous expression of the neuroendocrine marker Synaptophysin within ductal lesions. Higher percentages of Cytokeratin-Synaptophysin dual positive tumor cells correlate with shortened disease-free survival. We observe similar lineage marker heterogeneity in mouse models of pancreatic ductal adenocarcinoma, where lineage tracing indicates that Cytokeratin-Synaptophysin dual positive cells arise from the exocrine compartment. Mechanistically, MYC binding is enriched at neuroendocrine genes in mouse tumor cells and loss of MYC reduces ductal-neuroendocrine lineage heterogeneity, while deregulated MYC expression in KRAS mutant mice increases this phenotype. Neuroendocrine marker expression is associated with chemoresistance and reducing MYC levels decreases gemcitabine-induced neuroendocrine marker expression and increases chemosensitivity. Altogether, we demonstrate that MYC facilitates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma, contributing to poor survival and chemoresistance., Neuroendocrine differentiation of epithelial tumor cells can contribute to cancer cell resistance and survival. Here, the authors show that dysregulated c-Myc promotes neuroendocrine differentiation in pancreatic ductal adenocarcinoma, leading to poor survival and chemoresistance.

Aberrant activation of embryonic signaling pathways is frequent in pancreatic ductal adenocarcinoma (PDA), making developmental regulators therapeutically attractive. Here we demonstrate diverse functions for pancreatic and duodenal homeobox 1 (PDX1), a transcription factor indispensable for pancreas development, in the progression from normal exocrine cells to metastatic PDA. We identify a critical role for PDX1 in maintaining acinar cell identity, thus resisting the formation of pancreatic intraepithelial neoplasia (PanIN)-derived PDA. Upon neoplastic transformation, the role of PDX1 changes from tumor-suppressive to oncogenic. Interestingly, subsets of malignant cells lose PDX1 expression while undergoing epithelial-to-mesenchymal transition (EMT), and PDX1 loss is associated with poor outcome. This stage-specific functionality arises from profound shifts in PDX1 chromatin occupancy from acinar cells to PDA. In summary, we report distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. These findings provide insight into the complexity of PDA pathogenesis and advocate a rigorous investigation of therapeutically tractable targets at distinct phases of PDA development and progression.

Pancreatic ductal adenocarcinoma (PDA) is the second most common cause of cancer death in the US with a 10% 5-year survival rate and, as such, is one of the most common deadly cancers. PDA is characterized by a robust stromal reaction, a large portion of which is composed of infiltrating immune cells. This inflammatory response has been previously shown to promote both the initiation and progression of PDA. Current research has been to understand the interaction between tumor cells and infiltrating immune cells and the contribution to cancer progression. In this work we focus on a specific cell population present in the epithelium during pancreatic tumorigenesis called a tuft cell. Tuft cells have been found in luminal surfaces throughout the body, most notably characterized in the intestine where these cells have been shown to directly regulate the immune response to parasitic infection, but have also been found in the nasal, respiratory and bladder epithelium. These solitary chemosensory cells, as they are also known, use a common chemosensory pathway, previously characterized in type-II taste cells, in order to interact with the luminal environment. Interestingly, tuft cells in the metaplastic pancreas uniquely express TRPM5 and alpha-gustducin, both components of the chemosensory cascade, suggesting these cells have the potential to mediate a response to extracellular signals. Pancreatic tuft cells also make an array of cytokines and immunomodulatory factors that can directly alter the immune response in the tumor microenvironment. These data leads us to hypothesize that tuft cells utilize the chemosensory response pathway to remodel the tumor microenvironment in PDA. In order to answer this question, we ablated a key protein in the chemosensory cascade, alpha-gustducin, and found that this loss increases aggressiveness in a model of pancreatitis-associated tumorigenesis. We are currently exploring differences in the tumor microenvironment that may explain these results. Citation Format: Megan T. Hoffman, Howard Crawford. Tuft cell chemosensory signaling during pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1004.